gels Review Modeling the Mechanobiology of Cancer Cell Migration Using 3D Biomimetic Hydrogels Xabier Morales †, Iván Cortés-Domínguez † and Carlos Ortiz-de-Solorzano * IDISNA, Ciberonc and Solid Tumors and Biomarkers Program, Center for Applied Medical Research, University of Navarra, 31008 Pamplona, Spain; [email protected] (X.M.); [email protected] (I.C.-D.) * Correspondence: [email protected] † These authors contributed equally to this work. Abstract: Understanding how cancer cells migrate, and how this migration is affected by the me- chanical and chemical composition of the extracellular matrix (ECM) is critical to investigate and possibly interfere with the metastatic process, which is responsible for most cancer-related deaths. In this article we review the state of the art about the use of hydrogel-based three-dimensional (3D) scaffolds as artificial platforms to model the mechanobiology of cancer cell migration. We start by briefly reviewing the concept and composition of the extracellular matrix (ECM) and the materials commonly used to recreate the cancerous ECM. Then we summarize the most relevant knowledge about the mechanobiology of cancer cell migration that has been obtained using 3D hydrogel scaf- folds, and relate those discoveries to what has been observed in the clinical management of solid tumors. Finally, we review some recent methodological developments, specifically the use of novel bioprinting techniques and microfluidics to create realistic hydrogel-based models of the cancer ECM, and some of their applications in the context of the study of cancer cell migration. Keywords: hydrogel; collagen; Matrigel; extracellular matrix; mechanobiology; amoeboid-mesenchymal transition; cancer; cell migration; microfluidic devices; bioprinting Citation: Morales, X.; Cortés-Domínguez, I.; Ortiz-de-Solorzano, C. Modeling the Mechanobiology of Cancer Cell 1. Introduction Migration Using 3D Biomimetic Hydrogels. Gels 2021, 7, 17. https:// Cell migration is crucial for several physiological processes as diverse as tissue mor- doi.org/10.3390/gels7010017 phogenesis, immune cell trafficking, wound repair, and metastasis, one of the hallmarks of cancer malignancy [1,2]. Cell assays based on two-dimensional (2D) cellular models, Received: 31 December 2020 such as wound healing or scratch-based assays, are still widely used for migration research. Accepted: 9 February 2021 Therefore, most basic concepts about cell migration have been described from the study Published: 12 February 2021 of cell motility on top of 2D substrates made of one or several extracellular matrix (ECM) components [3,4]. In particular, the effect of relevant environmental factors, such as the Publisher’s Note: MDPI stays neutral ECM composition, the diffusion of chemical factors, or the topology and mechanical prop- with regard to jurisdictional claims in erties of the substrate in how cells migrate has been mostly analyzed in 2D [5] even if 2D published maps and institutional affil- systems cannot faithfully recapitulate the molecular and biomechanical complexity of 3D iations. in vivo environments. Indeed, there are specific characteristics of 3D environments that 2D models are not able to replicate, such as the cell’s spatial confinement, or cell–cell and cell–matrix interactions that affect proliferation, differentiation or the response to migration stimuli [6,7]. These limitations of 2D cellular models have fostered the development of Copyright: © 2021 by the authors. hydrogel-based 3D cellular models that more faithfully replicate the native environment of Licensee MDPI, Basel, Switzerland. migrating cells. This article is an open access article In this review, we start by reminding the reader of the concept and principal elements distributed under the terms and of the extracellular matrix of tissues. Then we review the state of the art on the materials conditions of the Creative Commons commonly used to fabricate hydrogels that mimic the composition and architecture of Attribution (CC BY) license (https:// normal and cancer tissues, and in that context, review the latest research on the mechanobi- creativecommons.org/licenses/by/ ology of 3D cancer cell migration. Finally, we present the latest engineering developments 4.0/). Gels 2021, 7, 17. https://doi.org/10.3390/gels7010017 https://www.mdpi.com/journal/gels Gels 2021, 7, 17 2 of 33 in bioprinting and the use of microfluidic devices to create realistic 3D environments to efficiently study cancer cell migration. 2. The Extracellular Matrix (ECM) The extracellular matrix is a highly organized protein structure that provides bio- chemical homeostasis and structural support to cells, tissues, and organs. The ECM is made of a complex network of cell-secreted macromolecules, including fibrous proteins, glycoproteins, and proteoglycans (PGs) [8,9] (Figure1). The relative abundance and spatial organization of these ECM constituents confer each tissue type with unique physical and biochemical properties [5,10]. These properties, e.g., the rigidity of the matrix or its porosity, affect cell behavior and actively contribute to homeostasis and tissue disease [11]. Figure 1. Extracellular matrix (ECM) composition and functions. The ECM is a non-cellular three-dimensional structure made of a large number of cell-secreted macromolecules that provide structural and biochemical support to surrounding cells. Structurally, ECM can be briefly summarized as a blend of water, fibrous proteins, and polysaccharides, e.g., proteoglycans (PGs) and glycosaminoglycans (GAGs). Collagen is the main structural component of the ECM that provides tensile strength to tissues, regulates cell adhesion by anchoring to integrins, and triggers cell differentiation and survival cues. Besides, the collagen lattice interacts with other non-collagenous glycoproteins, e.g., fibronectin, laminin, or elastin- favoring the reinforcement and spatial organization of the ECM. Fibronectin and laminin fibers play a pivotal role in ECM assembly, acting as “adhesive” proteins. Namely, these proteins allow the simultaneous binding to cell-surface receptors, e.g., integrins and syndecans or the cortical glycocalyx-, fibrillar proteins, and other focal adhesion molecules via multiple domains interspersed throughout their structure, which in turn influences cell proliferation, differentiation, and motility. Elastin fibers provide mechanical resilience and elasticity to tissues. Therefore, the collagen/fibronectin ratio confers unique mechanical properties to the tissues that allow both reversible extensibility behavior and the strength to bearing forces. On the other hand, PGs and GAGs fill the interstitial spaces forming a highly hydrated gel by sequestering water molecules, providing compressive strength and buffering properties to tissues. Furthermore, GAGs are a reservoir of growth factors, e.g., TGF-β, EGF, PDGF, etc., that trigger a wide range of fundamental physiological processes ranging from cell proliferation and differentiation to cell adhesion and motility. Indeed, GAGs also modulate cell behavior by interacting with cell-surface receptors to induce cytoskeleton-mediated mechanotransduction of signals and subsequent gene transcription. Collagen is the major structural constituent of the ECM. Collagen fibers self-organize into 3D networks whose density and tensile strength play a key role in cell migration and adhesion, affecting the maintenance of normal tissue physiology or the onset of pathological tissue behavior [10,12]. For instance, in the context of cancer research, the aberrant expression, deposition, alignment or cross-linking of various collagen subtypes Gels 2021, 7, 17 3 of 33 have been associated with the occurrence of mesenchymal–epithelial transition (MET), tumor dissemination, or drug resistance [13]. Non-collagenous glycoproteins, such as fibronectin, laminin, and elastin, are adhesive proteins extensively expressed in the ECM. These proteins, interspersed between the collagen fibers, increase the structural integrity of the mesh and participate in cell–matrix interactions and cell signaling, through binding with integrins and other cell surface receptors, such as syndecans or a bulky glycocalyx [14,15]. PGs and glycosaminoglycans (GAGs) form another relevant family of the ECM net- work. PGs form a gel-like, amorphous material that fills the interstitial spaces of the ECM, embedding other scaffolding proteins. Both PGs and GAGs constitute also a reservoir of bioactive molecules and growth factors, e.g., TGF-β or EGF, that regulate fundamental cell processes such as cell proliferation, migration, and differentiation [16]. Bearing in mind the complexity of the ECM composition, it is not surprising that alterations in one or a few constituents of the ECM may elicit remarkable changes in its biochemical and physical properties, leading to dysregulated cell behavior and disease. Accordingly, in vitro studies of disease must, to the largest possible extent, be based in models that replicate the biomechanical properties and the composition of the target ECM, in order to be of biological significance. 3. Three-Dimensional (3D) ECM-Mimicking Scaffolds: Materials Two-dimensional (2D) cellular models are being replaced by 3D hydrogel-based cell culture models [17,18]. Hydrogels are highly hydrated 3D polymeric scaffolds made of one or several physically or chemically cross-linked ECM-derived proteins: collagen derivatives, glycoproteins, polysaccharides such as hyaluronic acid (HA), or reconstituted cell-derived matrices (CDM) such as Matrigel©